An in vitro long-term study of cryopreserved umbilical cord blood-derived platelet-rich plasma containing growth factors-PDGF-BB, TGF-ß, and VEGF.

PURPOSE: Umbilical cord blood-derived platelet-rich plasma (UCB-PRP) containing growth factors has attracted attention as a biomaterial useful for regenerative medicine. The osteoblastic differentiation of umbilical cord-derived mesenchymal stromal cells (UC-MSCs) can be induced by UCB-PRP. MATERIALS AND METHODS: Nine samples of UC and UCB were used to conduct an in vitro study that determined the contents of three growth factors (i.e., platelet-derived growth factor, transforming growth factor ß-1, and vascular endothelial growth factor) and that examined, by staining with Alizarin red, their ability to induce the osteoblastic differentiation of UC-MSCs at the baseline, 3 months, and 3 years of cryopreservation. RESULTS: The contents of growth factors in cryopreserved UCB-PRP were markedly elevated compared to those found in UCB at baseline. The samples of UCB that were added with cryopreserved UCB-PRP and those with bone morphogenetic protein-2 were stained granularly with Alizarin red, thus indicating the presence of calcium. The samples of UCB that were not added with UCB-PRP were not stained with Alizarin red. The above-mentioned contents and ability were maintained at 3 years of cryopreservation. Cryopreserved UCB-PRP possibly and advantageously induced the osteoblastic differentiation of UC-MSCs. CONCLUSION: The potential clinical application of cryopreserved UCB-PRP to regenerative medicine was suggested.

Five-year efficacy of finasteride in 801 Japanese men with androgenetic alopecia.

Finasteride is standard medical treatment for androgenetic alopecia; however, no large studies with 5 years or more of follow up have been performed in Japan. The authors followed Japanese men with androgenetic alopecia treated with finasteride for 5 years to evaluate long-term treatment efficacy. Of 903 men treated with finasteride (1 mg/day), 801 patients were evaluated over 5 years by modified global photographic assessment. Although the proportion of improvement was high (99.4%), modified global photographic assessment scores after 5 years of treatment were lower in patients with more advanced disease as measured by the modified Norwood-Hamilton scale. After separating patients into "sufficient" and "insufficient" efficacy groups according to the modified global photographic assessment score after 5 years (scores ≥6 and <6, respectively), multivariate analysis showed that independent risk factors of insufficient efficacy were age at start of treatment of 40 years or more (P = 0.021) and classification on the modified Norwood-Hamilton scale (P < 0.001), whereas presence of stress at start of treatment was a negative predictor (P = 0.025). In conclusion, continuous finasteride treatment for 5 years improved androgenetic alopecia with sustained effect among Japanese. Younger age and less advanced disease at start of treatment were the key predictors of higher finasteride efficacy.

Single-stage reconstruction of a full-thickness alar defect using a folded nasolabial flap combined with a redundant skin turnover flap.

BACKGROUND: The reconstructive strategy for full-thickness nasal skin defects should include recreation of a cutaneous cover, support, and internal nasal lining. The most challenging aspect of this procedure is provision of the nasal lining. These reconstructions typically require a 2-step process. Satisfactory nasal skin reconstruction in a single operation is ideal. OBJECTIVE: We used a folded nasolabial flap combined with a turnover flap for reconstruction of full-thickness alar defects. METHODS: The donor material of the lining flap was a combination of the distal portion of the nasolabial flap and redundant skin resected during its transposition. The redundant skin flap was turned upside down, with the skin surface inside the nasal cavity. The remaining portion of the defect was covered with a folded nasolabial flap. RESULTS: This procedure was successful in all 5 patients. All flaps survived completely without evidence of necrosis or narrowing of airways. Aesthetic concerns, including effacement of the nasofacial sulcus, were minor. CONCLUSION: This method has the advantage of providing well-vascularized tissue of appropriate color, texture, and thickness for external coverage, as well as a satisfactory internal lining in a single-stage procedure.

Immediate reconstruction after removal of nasal prosthesis using diced cartilage wrapped with temporal fascia.

Various materials are used for nasal augmentations. Silicone is the most prevalent because it is durable and facilitates sculpturing. However, the unfortunate patient who presents with complication of the nasal implants and wants to remove them is vexed with a significant resultant cosmetic defect if the implant is removed. However, the patients who have some troubles after augmentation by the implants tend to hate the use of the prosthesis again. Ideally, immediate reconstruction would be offered to the patient, sparing him/her the deformity left by removal of the implant. We treated 16 patients who had undergone immediate nasal reconstruction after removal of foreign body. We reconstructed nasal deformity by diced cartilage wrapped with temporal fascia. The cartilage harvested from the ear concha was finally diced into 1- to 2-mm cubes. A bag was made from deep or superficial temporal fascia, and diced cartilage cubes were placed in the bag, which was grafted onto the nasal dorsum. This procedure had several advantages including getting natural contouring and enough volume and absence of foreign body reaction. It was also soft to the touch compared with prosthesis. The fascia could support the thin dorsum skin. The nasal augmentation effect of this procedure was comparable with that of prosthesis methods. It had lower risks for infection and exposure and provided more psychologic comfort. The nasal deformities were successfully reconstructed using diced cartilage wrapped with temporal fascia. We believe that this is the good method for the immediate nasal reconstruction after the removal of foreign body.

Significance of CD271 in bone marrow mesenchymal stem cells--changes by cryopreservation.

In investigating the biological activities of bone marrow mesenchymal stem cells (BMMSCs), an important task is cell sorting of effective BMMSCs. In the present study, we examined the usefulness of CD271 as a cell surface marker of BMMSCs. Specifically, we investigated (1) CD271 expression before and after freezing, (2) difference between the CD271(+) and CD271(-) fractions regarding calcium formation level after bone differentiation, and (3) method of harvesting effective BMMSCs from marrow tissue. CD271 was partially expressed in cryopreserved BMMSCs (CBMMSCs). We used CD271 in separating CBMMSCs into different cell groups and compared the calcium formation levels between the CD271-expressed and CD271-nonexpressed groups. A significant amount of BMMSCs existed even in the CD271(-) fraction, and the calcium formation level was high in 4 of 5 CD271(-) fraction specimens. The same investigation was conducted on nonfrozen BMMSCs. No major difference was found in CD271 expression compared with CBMMSCs. However, the calcium formation level of the CD271(+) fraction was higher in 3 of 5 specimens. We presumed that CD271 expression might have been substantially changed during culture and cryopreservation. We compared the method of directly culturing bone marrow tissue and that of washing the sample before culture and confirmed that the calcium formation level of BMMSCs was higher when the marrow tissue was washed before culture.

[Allogeneic bone marrow transplantation for chemotherapy-resistant T-prolymphocytic leukemia].

A 34-year-old female was referred to our hospital for the evaluation of atypical lymphocytosis. Leukocyte count at diagnosis was 17,900/microl with 58% atypical lymphocytes having a convoluted nucleus and prominent nucleoli. Because the leukocyte count increased to 43,600/microl, the patient was treated with 2'deoxycoformycin followed by CHOP combination chemotherapy. However, both treatments failed to achieve remission. We planned an allogeneic bone marrow transplantation from an HLA-matched unrelated donor. The patient was treated with Ara-C and etoposide before conditioning to decrease the high leukemia burden. After administration of total body irradiation (12 Gy in six fractions) and cyclophosphamide (total dose of 120 mg/kg) unmanipulated marrow cells were infused. Under prevention of GVHD by CsA and short-term MTX, leukocyte engraft was prompt at day 16, and acute GVHD grade II was observed. Because 9.4% of residual recipient type T-cells was seen with STR analysis on day 22, we decreased the dose of Cs'A. After the occurrence of mild acute GVHD, the residual T-cell number decreased. The patient is still in complete remission for up to 22 months after BMT. We conclude that allogeneic SCT is effective for the treatment of T-PLL.

Experimental study on bone formation potential of cryopreserved human bone marrow mesenchymal cell/hydroxyapatite complex in the presence of recombinant human bone morphogenetic protein-2.

Secondary bone grafting in the alveolar cleft is one of the most important treatment modalities for patients with a cleft lip and palate. In children, however, harvesting a sufficient amount of bone graft from the donor site is difficult, and the procedure imposes a heavy burden on the patients. This problem may be resolved if autologous transplantation can be performed using a cell-hybrid type of artificial bone prepared with the patient's own bone marrow cells through a tissue engineering technique. In the current study, we examined the possibility of achieving such an autologous transplantation using cryopreserved human bone marrow cells. Human bone marrow cells were grown in culture and cryopreserved, and the cells preserved for 3 years and the cells preserved for 3 months were then thawed and recultivated. In one experiment, the recultivated cells were seeded onto a complex composed of porous hydroxyapatite and bone morphogenetic protein (BMP), and the complex was grafted subcutaneously in nude mice. In another experiment, the cells were seeded onto the porous hydroxyapatite and cultivated for 10 days before grafting in a medium supplemented with BMP. The bone formation potential of the cells was compared between these two experiments. Formation of mature bone was observed 2 weeks after transplantation in the former experiment, whereas only scarce bone formation was evident 4 weeks after transplantation in the latter experiment. It is therefore assumed that exposing the cultured human bone marrow cells to a high concentration of BMP in vivo strongly promotes bone formation.

The generation of immunocompetent dendritic cells from CD34+ acute myeloid or lymphoid leukemia cells.

The ability of CD34+ leukemic cells to differentiate to dendritic cells (DCs) was investigated in 18 acute myeloid leukemia (AML) and 4 lymphoid leukemia (ALL) patients. The generation of DCs was determined by the expression of DC-associated CD1a or CD83 (more than 30%) with costimulatory molecules, by CD80 antigens (>20%), and by the exhibition of allostimulatory activity. In the AML patients, allostimulatory mature DCs were generated from 3 of 9 M0 or M1, 2 of 5 M2,2 of 4 M4 or M5, and 3 of 4 ALL (L2) cases. In total, DCs were more efficiently induced from cases expressing over 75% of CD34+ among whole bone marrow mononuclear cells (8 of 12), compared with those under 75% (2 of 10; P < .05). B-cell (CD19), natural killer (NK)-cell (CD56), or T-cell (CD7) lineage markers, which were aberrantly expressed on the blasts, were rarely found on leukemic DCs at the end of the culture period, and myeloid (CD13, CD33), not lymphoid (CD10), markers were shown on ALL-derived DCs. In Philadelphia chromosome-positive ALL or AML patients with t (8;21), DCs were confirmed to be of leukemic origin by fluorescence in situ hybridization analysis.